Fungal infections are common in the United States, and are often associated with significant morbidity. For example, Candida species are the fourth most common organisms isolated from US hospital patients (Wenzel and Pfaller, Infect. Control Hosp. Epidemiol., 12:523-524 (1991)). Histoplasma capsulatum and Coccidioides immitis causes, respectively, 250,000 and 100,000 new infections annually, and 10-40% of infected persons become symptomatic (Bullock, Histoplasma capsulatum, In: Principles and Practice of Infectious Diseases, G L Mandell et al. (eds.), Churchill Livingstone, N.Y., 1995, pp. 2340-2353 and 2365-2375; and Stevens, N. Engl. J. Med., 332:1077-1082 (1995)). Individuals with symptomatic C. immitis infection miss an average of 35 days of school or work, and incur a total of $24 million in medical expenses annually.
Many fungi cause respiratory infections that are indistinguishable on the basis of symptoms. However, it is important to accurately determine which fungus is responsible for infection due to differences in treatment regimens and incidences of complications (Bullock, Histoplasma capsulatum, In: Principles and Practice of Infectious Diseases, G L Mandell et al. (eds.), Churchill Livingstone, N.Y., 1995, pp. 2340-2353 and 2365-2375; and Galgiani, Ann. Intern. Med., 130:293-300 (1999)). Because culture and direct antigen detection are problematic, antibody detection plays an important role in the diagnosis and identification of fungal infections. The methods most commonly used to detect fungal antibodies include complement fixation (CF) and immunodiffusion (ID) (Zancope-Oliveira et al., Clin. Diagn. Lab. Immunol., 1:90-93 (1994)). Both methods, however, have inherent drawbacks. ID detects antibodies to species-specific protein moieties, and is thus sensitive and highly specific; however, the assay takes 48 hours to perform, and appropriate interpretation requires highly skilled personnel. Although CF assays are highly sensitive, their performance is complex and labor-intensive, and they exhibit low specificity due to cross-reactive antibodies recognizing carbohydrate moieties common to several fungi (Zancope-Oliveira et al., Clin. Diagn. Lab. Immunol., 1:90-93 (1994); and Yang et al., Clin. Diagn. Lab. Immunol., 4:19-22 (1997)). Enzyme-linked immunosorbent assays (ELISA) for the detection of fungal antibodies have also been described, but these assays exhibit low specificity due to the same cross-reactive antibodies at issue in CF assays (Zartarian et al., Am. J. Clin. Pathol., 207:148-153 (1997); and Kaufman et al., J. Clin. Microbiol., 33:618-619 (1995)).
During the last decade, investigators using a western blot assay system found that periodate oxidation of fungal antigen preparations inactivates the cross-reactive carbohydrate moieties but does not disturb the structural integrity of the protein moieties (Zancope-Oliveira et al., Clin. Diagn. Lab. Immunol, 1:90-93 (1994); Zancope-Oliveira et al., Clin. Diagn. Lab. Immunol., 1:563-568 (1994); and Pizzini et al., Clin. Diagn. Lab. Immunol., 6:20-23 (1999)). Thus, utilization of oxidized fungal antigens markedly increased the specificity of the western blot assay for fungal antibodies, without affecting sensitivity. However, the western blot assay described therein is not practical for most medical laboratories; many complex steps are required, including antigen preparation, gel electrophoresis, electrotransfer to nitrocellulose membranes, assay performance, and blot interpretation. The antigen was a culture filtrate, 0.45 μm filtered, 20× concentrated by ultrafiltration, dialyzed against PBS, purified by ion exchange chromatography, 10× concentrated by ultrafiltration, and finally dialyzed against PBS. Oxidizing the antigen also included several labor intensive and potentially contaminating steps: 4 mg/mL antigen was exposed to periodate for 18 hours at 4° C., treated with glycerol for 15 minutes, treated with borohydrate for 2 hours, and finally dialyzed against water. Manufacturing the devices also included methods requiring sophisticated equipment and skilled technicians: denaturing the oxidized antigen with heat and mercaptoethanol, electrophoresing with a 4% stacking gel and 7.5% resolving gel, transferring the proteins to nitrocellulose paper, blocking with buffered milk and tween, drying the paper, and cutting the paper into strips, etc.
Although attempts to utilize periodate-treated fungal antigens for specific detection of fungal antibodies in an ELISA system have been reported, these attempts were only marginally successful (Fisher et al., Mycroses, 40:83-90 (1997)). Binding of H. capsulatum antibodies to oxidized Blastomyces dermatitidis antigens was reduced only by about 20% when compared to binding to non-oxidized B. dermatitidis antigens. This limited reduction in cross-reactivity may have been due to the use of highly purified (and thus extensively manipulated) fungal antigens, periodate oxidation after the antigens had adsorbed to the plastic surface, or the use of an insufficient concentration of periodate.
Therefore, there exists a need in the art for oxidized fungal antigens with more reduced cross-reactivity. The present invention addresses this and other related needs.